Polymeric systems for the delivery of anticancer drugs

ABSTRACT

The present invention relates to compositions for the treatment of cancerous tissues in warm-blooded animals containing one or two anticancer agents attached to polymeric carriers having monomer units derived from one or more of N-(2-carboxypropyl)methacrylamide (2-CPMA), N-(3-carboxypropyl)methacrylamide (3-CPMA), N-(2-aminopropyl)methacrylamide (2-APMA) and/or N-(3-aminopropyl)methacrylamide (3-APMA) are also included. Anticancer agents in compositions can be attached to said polymeric carrier by side-chains which can be susceptible to hydrolysis by lysosomal enzymes intracellularly. Compositions can also include a targeting ligand attached to the polymeric carrier, optionally through a second linker.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/903,927, filed on Oct. 13, 2010, and claims priority to U.S.Provisional Application No. 61/251,156 filed Oct. 13, 2009, the entirecontents of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention includes compositions containing one or twoanticancer agents attached to polymeric carriers for the treatment ofcancerous tissues in warm-blooded animals. The polymeric carriersinclude functional groups such as the amino group or carboxylic acidgroup. One or two anticancer agents in compositions may be attached tosaid polymeric carrier by side-chains which are susceptible tointracellular hydrolysis by lysosomal enzymes and wherein said polymericcarriers optionally contain a targeting ligand.

BACKGROUND OF THE INVENTION

Many low molecular weight drugs used in chemotherapy rapidly enter alltypes of cells by random diffusion through the cell membrane. This lackof selectivity decreases availability at the desired target cells ortissue and sometimes causes undesirable side effects. Cellular uptake israpid so that the therapeutic effect is not extended over a period oftime. Furthermore, glomerular filtration can rapidly remove the drugsfrom the bloodstream.

The covalent attachment of low molecular weight bioactive molecules tosoluble polymeric carriers both prevents glomerular filtration andpromotes cellular absorption by mechanisms other than just simplediffusion.

Synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer is anexample of a biocompatible, water-soluble, inert and neutral drugcarrier for in vivo delivery of anti-cancer therapeutics (See, forexample, U.S. Pat. Nos. 4,062,831 and 4,097,470). The conjugation ofanticancer drugs to HPMA copolymers results in many advantageousfeatures over small molecular therapeutics, including improvedsolubility and bioavailability, preferential accumulation of theconjugates in solid tumors or passive tumor targeting due to theenhanced permeability and retention (EPR) effect, reduced systemictoxicity and enhanced therapeutic efficacy, and down-regulation ofmulti-drug resistance.

There are currently six HPMA copolymer-drug conjugates at various stagesof clinical trials and their preparation and compositions are disclosedin several patents. There are currently two polyglutamate-drugconjugates at various stages of clinical trials, and furtherpolymer-drug conjugates including dextran-drug conjugates and PEG-drugconjugates are reported in clinical or preclinical development.

U.S. Pat. No. 5,037,883 issued Aug. 6, 1991, (Kopecek et al.) describesa drug conjugate of an inert polymeric carrier attached through apeptide linkage to bio-active molecules. This patent describescopolymers of N-(2-hydroxypropyl)methacrylamide containing oligopeptidesequences terminated in anticancer drugs (e.g. adriamycin, daunomycin,melphalan) and bound to targeting moieties (e.g. galactosamine,fucosylamine, anti-Thy 1.2 antibodies, anti-Ia antibodies) that arealleged to have a higher therapeutic efficacy compared to the lowmolecular weight drugs that contain no polymers. In particular aconjugate containing adriamycin as a drug (bound via a Gly-Phe-Leu-Glyoligopeptide sequence) and galactosamine as a targeting ligand isdescribed. U.S. Pat. No. 6,692,734 (Stewart etc., 2004) and U.S. Pat.No. 7,166,733 (Nowotnik, 2007) describe a poly(HPMA)-GFLG-platinum drug.Luo et al. in US 2004/0234497 discloses a cell-targeted polymericdelivery system of poly(HPMA)-GFLG-HA-doxorubicin that was designedbased on the specific interaction between hyaluronic acid (HA) and itscell surface receptors overexpressed on cancer cell surface. In US2006/0014695, Ghandehari et al., also describes compositions and methodsfor nucleic acid delivery comprising HPMA conjugated to a polyamine.Another issued patent, U.S. Pat. No. 5,258,453 (Kopecek and Krinick,1993) describes the combination effect of the anticancer agent andphotoactivatable drug by poly(HPMA)-GFLG-adriamycin-ce6-secretin.Lammers et al. showed the delivery of two different chemotherapeuticagents (doxorubicin and gemcitabine) to tumors simultaneously using HPMAcopolymers (Biomaterials 30: 3466-3475 (2009)).

There remains a need for additional polymer carriers for the delivery ofanticancer drugs and the like.

SUMMARY OF THE INVENTION

Embodiments include therapeutic compositions having a first polymercarrier. The first polymer carrier has a first monomer, which isN-(2-carboxypropyl)methacrylamide (2-CPMA),N-(2-carboxypropyl)methacrylamide (3-CPMA),N-(3-aminopropyl)methacrylamide (3-APMA), orN-(2-aminopropyl)methacrylamide (2-APMA). The therapeutic compositionsfurther include a first anticancer agent attached to the first polymercarrier, optionally through a linker; where the first anticancer agentis attached to the first monomer or to another monomer. For example, thepolymer carrier may be a homopolymer of 2-CPMA, 3-CPMA, 3-APMA, or2-APMA where at least some of the monomer units are chemically attachedto an anticancer agent.

In some embodiments, the first polymer carrier has a second monomer. Inother words, the first polymer carrier is a copolymer. For example, thepolymer carrier is a copolymer having 2-CPMA, 3-CPMA, 3-APMA, and/or2-APMA monomers. In the case of copolymers, at least some of the 2-CPMA,3-CPMA, 3-APMA, or 2-APMA may be chemically attached to an anticanceragent. In some embodiments, the first anticancer agent is attached tothe second monomer, optionally through a linker. For example, in thesecopolymers, the 2-CPMA, 3-CPMA, 3-APMA, or 2-APMA monomers may beunmodified, and the copolymer includes a monomer other than 2-CPMA,3-CPMA, 3-APMA, or 2-APMA that is chemically attached to the anticanceragent. In some embodiments, the second monomer isN-(2-hydroxypropyemethacrylamide (HPMA), an acrylamide, amethacrylamide, an acrylate or a methacrylate. These monomers may beunderivatized or derivatized to be chemically attached to, for example,one or more anticancer agents or targeting ligands, or linkers.

In some embodiments, the composition further includes an additionalanticancer agent, different from the first anticancer agent. Theadditional anticancer agent is attached to the first polymer carrier,optionally through a linker. The additional anticancer agent may beattached to the first monomer or to another monomer. In someembodiments, both anticancer agents are attached to the first type ofmonomer (i.e. 2-CPMA, 3-CPMA, 2-APMA, or 3-APMA), meaning that at leastsome individual units of the first monomer have been chemically modifiedwith each anticancer agent. In some embodiments, one anticancer agent isattached to the first monomer, while the other is attached to a monomerother than the first monomer. In some embodiments, both anticanceragents are attached to monomers other than the first monomer. In anycase, the monomers to which the two anticancer agents are attached maybe the same or different.

In some embodiments, the composition further includes a targeting ligandattached to the first polymer carrier, optionally through a linker. Asabove, the targeting ligand may be attached to the first monomer or toanother monomer. As above, the targeting ligand may be attached to thesame type of monomer (either the first or another monomer) as the firstanticancer agent or the second anticancer agent (if present). In otherexample, the targeting ligand is attached to a different type of monomerthan the first or second anticancer agent. In some embodiments, thetargeting ligand may be RGDfK, EPPT1, or folate.

Embodiments include compositions that further include a second polymercarrier and a second anticancer agent and/or a targeting ligand attachedto to the second polymer carrier, optionally through a linker. In someembodiments, second polymer carrier is a polymer comprising a monomerselected from N-(2-hydroxypropyl)methacrylamide (HPMA),N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(3-aminopropyl)methacrylamide (3-APMA), andN-(2-aminopropyl)methacrylamide (2-APMA).

In any embodiment, the first anticancer agent may be attached to thefirst polymer carrier through a linker. Likewise, where there aremultiple anticancer agents and/or targeting ligands attached to apolymer carrier, any or all of them may be attached through a linker. Inembodiments having at least two anticancer agents attached to onepolymer carrier, both are attached by linkers. In embodiments havingmore than one linker, each linker may be the same or different.

In some embodiments where at least one anticancer agent is attached tothe polymer through a linker, one or more or all of the linkers may besusceptible to cleavage by lysosomal enzymes. Examples of linkerssusceptible to cleavage by lysosomal enzymes include oligopeptidesequences, oligosaccharide sequences and structures similar to those innucleic acids. In some embodiments, the linker is an oligopeptidesequence. Examples of oligopeptide sequences include Gly-Gly,Gly-Phe-Gly, Gly-Phe-Phe, Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala,Gly-Leu-Phe, Gly-Leu-Ala, Ala-Val-Ala, Gly-Phe-Leu-Gly (SEQ ID NO: 1),Gly-Phe-Phe-Leu (SEQ ID NO: 2), Gly-Leu-Leu-Gly (SEQ ID NO: 3),Gly-Phe-Tyr-Ala (SEQ ID NO: 4), Gly-Phe-Gly-Phe (SEQ ID NO: 5),Ala-Gly-Val-Phe (SEQ ID NO: 6), Gly-Phe-Phe-Gly (SEQ ID NO: 7),Gly-Phe-Leu-Gly-Phe (SEQ ID NO: 8), or Gly-Gly-Phe-Leu-Gly-Phe (SEQ IDNO: 9). In some embodiments, the oligopeptide linker is Gly-Phe-Leu-Gly(SEQ ID NO: 1). In embodiments having more than one linker, a secondlinker may be an amino acid or peptide. In some embodiments, the secondlinker may be Gly-Gly (GG).

In some embodiments, the first anticancer agent may be docetaxel,gemcitabine, cisplatin, derivatives of docetaxel, derivatives ofgemcitabine, or derivative of cisplatin. In embodiments having at leasttwo anticancer agents, at least one of the anticancer agents may bedocetaxel, gemcitabine, cisplatin, a derivative of docetaxel, aderivative of gemcitabine, or a derivative of cisplatin. In someembodiments having at least two anticancer agents, one may be docetaxel,or a derivative of docetaxel, and the other may be gemcitabine or aderivative of gemcitabine. In some embodiments, having two anticanceragents, the first anticancer agent is docetaxel or a derivative ofdocetaxel, and the second or additional anticancer agent is cisplatin ora derivative of cisplatin.

Embodiments include pharmaceutical compositions including one or more ofthe above described compositions. Other embodiments include methods oftreating neoplastic diseases by administering a therapeuticallyeffective amount of one of the above described compositions. Otherembodiments include the use of one of the above described compositionsto treat a neoplastic disease.

Compositions according to the invention can include the monomer,N-2-carboxypropyl methacrylamide (2-CPMA) or N-3-carboxypropylmethacrylamide (3-CPMA) which have a carboxylic acid group rather thanthe neutral hydroxyl group as in HPMA. The CPMA monomers 2-CPMA and3-CPMA are used to synthesize CPMA based copolymers for the delivery ofanticancer agents. The carboxylic acid group has advantages over HPMA inthat it can be used for the production of esters, acid halides, acidamides, and acid anhydrides. In addition, the carboxylic acid in thisacidic drug carrier can be used for the conjugation of peptides, drugs,or polymers containing other functional group to this carboxylic group.It can be used before polymerization or after polymerization to extendor add the polymeric chains.

Embodiments of the invention include acidic drug carriers, such as2-CPMA-containing or 3-CPMA-containing polymers and copolymers, andbasic drug carriers, such as N2-APMA-containing or 3-APMA-containingpolymers and copolymess that are useful polymeric systems for thedelivery of one kind of anticancer agent or two kinds of differentanticancer agents.

Embodiments of the invention also relate to new polymeric systems todeliver anticancer agents for the treatment of neoplastic diseases. Thesystems are composed of a water-soluble polymer with one or twodifferent anticancer agents attached to the polymer backbone, forexample, through peptide linkages.

Embodiments of the invention relate to conjugates of anticancer agentsand/or targeting ligands to water-soluble polymers, such as polyN-(2-carboxypropyl)methacrylamide (p2-CPMA), polyN-(3-carboxypropyl)methacrylamide (p3-CPMA), polyN-(3-aminopropyl)methacrylamide (p3-APMA), or polyN-(2-aminopropyl)methacrylamide) (p2-APMA) and use of those conjugatesas specific intracellular carriers of anticancer agents into tumors.Examples of targeting ligands include RGDfK, EPPT1 peptide, or folate.

Embodiments of the present invention include soluble bioactive polymerssuch as poly N-(2-carboxypropyl)methacrylamide (p2-CPMA), polyN-(3-carboxypropyl)methacrylamide (p3-CPMA), polyN-(3-aminopropyl)methacrylamide (p3-APMA) and polyN-(2-aminopropyl)methacrylamide (p2-APMA) and related copolymerscontaining the same monomers and having pendant anticancer agents and apendant targeting ligand attached by enzymatically degradable bonds.

The present invention also provides a method for the treatment ofneoplastic diseases by the administration of soluble bioactivecopolymers, such as poly N-(2-carboxypropyl)methacrylamide (p2-CPMA),poly N-(3-carboxypropyl)methacrylamide (p3-CPMA), polyN-(2-aminopropyl)methacrylamide (p2-APMA) or polyN-(3-aminopropyl)methacrylamide (p3-APMA) and related copolymerscontaining the same monomers and containing pendant anticancer agents,optionally attached via enzymatically degradable bonds. The copolymermay also contain a targeting ligand specific for a tumor marker on thecancer cell.

The invention further provides a method for the treatment of neoplasticdisease by administration of copolymers containing two or more differentanticancer agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inhibition of tumor growth in nude micesubcutaneously injected with HCT116 human colon carcinoma cells bypHPMA-GFLG-Docetaxel (H1) and p2-CPMA-GFLG-Docetaxel (C1). ‘GFLG’ isdisclosed as SEQ ID NO: 1.

FIG. 2. shows schematic representations of example embodiments. FIG. 2Ashows the anticancer agent attached to the polymer carrier via acovalent bond. FIG. 2B shows a linker molecule directly attached to thepolymer carrier via a covalent bond, and the anticancer agent directlyattached to the linker molecule via a covalent bond. FIG. 2C shows ananticancer agent directly attached to the polymer carrier via a covalentbond, and a targeting ligand directly attached to the polymer carriervia a covalent bond. FIG. 2D shows a linker molecule directly attachedto the polymer carrier via a covalent bond, and the anticancer agentdirectly attached to the linker molecule, and a targeting liganddirectly attached to the polymer carrier via a covalent bond. FIG. 2Eshows a linker molecule directly attached to the polymer carrier via acovalent bond, and a targeting ligand directly attached to the linkermolecule, and an anticancer agent directly attached to the polymercarrier via a covalent bond. FIG. 2F shows a linker molecule directlyattached to the polymer carrier via a covalent bond, and the anticanceragent directly attached to the linker molecule, and a targeting liganddirectly attached to a different linker molecule, directly attached tothe polymer carrier via a covalent bond. FIG. 2G shows a compound havingtwo anticancer agents, each separated from the carrier by separatelinkers. FIG. 2H shows a compound having two anticancer agents withseparate linkers, and a targeting ligand, separated by a linker. FIG. 2Ishows a polymer carrier having attached thereto a targeting ligand. FIG.2J shows a polymer carrier having attached thereto a targeting ligandthrough a linker.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention are discussed in detail below. Indescribing embodiments, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected. A person skilled in the relevant artwill recognize that other equivalent parts can be employed and othermethods developed without parting from the spirit and scope of theinvention. All references cited herein are incorporated by reference asif each had been individually incorporated.

Free anticancer drugs diffuse throughout a cell and are not concentratedat a specific subcellular location. In addition, if such drugs areadministered intravenously they are systemically distributed to alltissues of the body. The action of these drugs at these unintendeddistribution sites results in observable systemic side effects. It isthus preferred to localize the drug to the sites in the body where theaction is desired. Targeting these agents to the subcellular site wherethey are most effective increases their efficacy and decreases theirtoxicity.

Targeting of anticancer drugs to tumors can be achieved by “passivetargeting” and “active targeting”. Passive targeting involves the use ofgenerally non-specific methods to selectively increase drug deliver totarget cells. For example, passive targeting can be achieved byincorporation or attachment of anticancer drugs into macromolecularcarriers such as water-soluble polymers. Active targeting utlilizesmoieties that are specific to recognition molecules (receptors) on thesurface of target cells. For example, active targeting can be achievedby attaching cellular targeting moieties to delivery systems such as amacromolecular carrier.

Polymers localize preferentially in solid tumors when compared to normaltissue. This occurs due to a phenomenon called the Enhanced Permeabilityand Retention (“EPR”) effect, which is attributed to morphologicalchanges in tumor tissue or cells, where the leaky vasculature produceddue to neoangiogenesis results in the leakage of vascular contents intothe extracellular tissue. In addition, the lymphatics may be blocked,which results in the accumulation of macromolecular agents in theextracellular tissue surrounding tumor cells. This phenomenon can beused to target tumor cells by attaching drugs to the polymers. Sincepolymers localize around tumor cells, the drugs attached to the polymersare also available at higher concentrations around the tumor. Anticanceragents attached to polymers are taken inside cells by endocytosis.Anticancer agents attached to polymers may retain their anticanceractivity. However, since the agents remain covalently attached to thepolymer backbone, in some instances they may not be as effective as freeagents. This may be overcome by the use of biodegradable or hydrolysablebonds or linkers, such as peptide sequences, to attach the drug to thepolymer backbone. When peptide sequences are used, the sequences arechosen such that they can be degraded inside the cell under specificconditions.

In addition, cancer cells often have surface molecules that are eitherabsent in normal tissue or over-expressed in comparison to the normaltissue. These may include growth factor receptors and/or certainantigens. Attaching recognition molecules to polymers that bind to thesemolecules results in selective binding of the carrier to tumor cells andtissue and a high concentration of polymers in the local environment ofthe tumor. Such targeting moieties include antibodies and peptidylligands for cell surface receptors. Receptor mediated endocytosisinitiated by the binding of some of these recognition molecules to theirreceptors may result in an increased intracellular concentration andcorrespondingly may result in an enhanced therapeutic effect.

Polymer-based therapeutics have a large hydrodynamic volume, whichtranslates into a longer intravascular half-life. Polymer-basedtherapeutics also enhance the solubility and the bioavailability ofinsoluble drugs. Other advantages afforded by polymer-based therapeuticsinclude increased maximum tolerated dose, decreased non-specifictoxicity, enhanced induction of apoptosis, and activation of alternatesignaling pathways (Kopecek et al., Advances in Polymer Science, 122(Biopolymers II): 55-123 (1995)).

Embodiments include new polymer based therapeutics and their use for thetreatment of neoplastic diseases. Embodiments include homopolymers andcopolymers produced from the polymerization of one or more ofN-(2-hydroxypropoyl)methacrylamide (HPMA),N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(2-aminopropyl)methacrylamide (2-APMA), orN-(3-aminopropyl)methacrylamide (3-APMA), where the homopolymer orcopolymers includes at least one of 2-CPMA, 3-CPMA, 2-APMA, or 3-APMA.

Monomers

As used herein, the terms monomor or comonomer may be used to mean amonomer prior to polymerization. However, the when used in reference toa formed polymer, the terms monomer, comonomer, monomer unit, orcomonomer unit refer to the subunit in a polymer or copolymer chainderived from a monomer or comonomer in its un-polymerized form. The useof these terms will be evident to persons skilled in the art in thecontext of the specification and claims.

As used herein, the terms “HPMA”, “2-CPMA”, “3-CPMA”, “2-APMA” and“3-APMA” mean the compounds N-(2-hydroxypropyl)methacrylamide,N-(2-carboxypropyl)methacrylamide, N-(3-carboxypropyl)methacrylamide(3-CPMA), N-(2-aminopropyl)methacrylamide, andN-(3-aminopropyl)methacrylamide, respectively as represented by thefollowing structure:

As used herein, an “underivatized” monomer is a monomer that has notbeen chemically modified. Underivatized monomers include, for example,2-CPMA, 3-CPMA, HPMA, 2-APMA, 3-APMA, acrylamide, methacrylamide,acryloyl chloride, methacryloyl chloride, acrylates, methacrylates, andother vinylic comonomers. A “derivatized” monomer means a monomer thathas been chemically modified (other than by polymerization). Thechemical modification may occur before or after polymerization. In someembodiments, a “derivatized” monomer is synthesized beforepolymerization. In some embodiments, a monomer or monomer unit is“derivatized” after polymerization. A monomer can be “derivatized” toattach, for example, an anticancer agent, a linker, a targeting ligand,a linker-anticancer agent combination, or a linker-targeting ligandcombination. Examples of derivatized monomers include 2-CPMA or 3-CPMA,where the carboxylic acid functional group is modified to be directlyattached, for example, by a covalent bond, to a linker, anticancer agentor targeting ligand or 2-APMA or 3-APMA, where the amine functionalgroup is modified to be directly attached, for example, by a covalentbond, to a linker, anticancer agent or targeting ligand. Other examplesinclude 2-CPMA or 3-CPMA where the carboxylic acid functional group isdirectly attached, for example, by a covalent bond, to a linker, wherean anticancer agent or targeting ligand is directly attached, forexample, by a covalent bond, to the linker. Other examples include2-APMA or 3-APMA where the amine functional group is directly attached,for example, by a covalent bond, to a linker, where an anticancer agentor targeting ligand is directly attached, for example, by a covalentbond, to the linker.

Polymer-Based Therapeutics

Embodiments of the invention include polymer based therapeutic compoundsthat can be used, for example, in anticancer therapies. These compoundsmay increase or alter the targeted delivery of anticancer compounds orother therapeutic compounds. Polymer based therapeutic compounds includehomopolymers and copolymers produced from the polymerization of one ormore of N-(2-hydroxypropoyl)methacrylamide (HPMA),N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(2-aminopropyl)methacrylamide (2-APMA), orN-(3-aminopropyl)methacrylamide (3-APMA), where the homopolymer orcopolymers includes at least one of 2-CPMA, 3-CPMA, 2-APMA, or 3-APMA,and where the homopolymer or copolymer includes at least one anticanceragent.

These polymer based therapeutic compounds comprise an anticancer agentand a polymer carrier. The polymer based therapeutic compounds mayoptionally comprise a linker, such as, for example, Gly-Phe-Leu-Gly (SEQID NO: 1), and/or a targeting ligand such as, for example, RGDfK, EPPT1peptide or folate. As used herein, a polymer carrier is the polymerbackbone attached to one or more anticancer agents, linkers, targetingligands, linker-anticancer agent combinations, or linker-targetingligand combinations.

Embodiments include therapeutic compositions having a first polymercarrier. The first polymer carrier has a first monomer, which is eitherN-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(3-aminopropyl)methacrylamide (3-APMA), orN-(2-aminopropyl)methacrylamide (2-APMA). The therapeutic compositionsfurther include a first anticancer agent attached to the first polymercarrier, optionally through a linker; where the first anticancer agentis attached to the first monomer or to another monomer. For example, thepolymer carrier may be a homopolymer of 2-CPMA, 3-CPMA, 3-APMA, or2-APMA where at least some of the monomer units are chemically attachedto an anticancer agent.

In some embodiments, the first polymer carrier has a second monomer. Inother words, the first polymer carrier is a copolymer, i.e., a copolymerhaving 2-CPMA, 3-CPMA, 3-APMA, and/or 2-APMA monomers. In the case ofcopolymers, at least some of the 2-CPMA, 3-CPMA, 3-APMA, or 2-APMAmonomer units may be chemically attached to an anticancer agent. In someembodiments, the first anticancer agent is attached to a second monomer,optionally through a linker. For example, in some copolymers, the2-CPMA, 3-CPMA, 3-APMA, or 2-APMA monomers may be unmodified, and thecopolymer includes a monomer other than 2-CPMA, 3-CPMA, 3-APMA, or2-APMA that is chemically attached to the anticancer agent. In someembodiments, the second monomer is N-(2-hydroxypropyl)methacrylamide(HPMA), an acrylamide, a methacrylamide, an acrylate or a methacrylate.These monomers may be underivatized or derivatized to be chemicallyattached to, for example, one or more anticancer agents or targetingligands, or linkers. Comonomers used in HPMA polymer and copolymerdelivery systems known in the art are example comonomers for use in thepresent invention.

In some embodiments, the composition further includes an additionalanticancer agent, different from the first anticancer agent. Theadditional anticancer agent is attached to the first polymer carrier,optionally through a linker. The additional anticancer agent may beattached to the first monomer or to another monomer. In someembodiments, both anticancer agents are attached to the first monomer,meaning that different units of the first monomer have been chemicallymodified with each anticancer agent. In such cases, some monomer unitsare attached to the first anticancer agent, and some to the secondanticancer agent, while some are underivatized. In some embodiments, oneanticancer agent is attached to the first monomer, while the other isattached to a monomer other than the first monomer. In some embodiments,both anticancer agents are attached to monomers other than the firstmonomer. In any case, the monomers to which the two anticancer agentsare attached may be the same or different.

In some embodiments, the composition further includes a targeting ligandattached to the first polymer carrier, optionally through a linker. Asabove, the targeting ligand may be attached to the first monomer or toanother monomer. As above, the targeting ligand may be attached to thesame monomer (either the first or another monomer) as the firstanticancer agent, or the second anticancer agent if present. In otherwords, the monomer attached to the first anticaner agent is of the sametype (i.e. 2-CPMA, 3-CPMA, 2-APMA, 3-APMA, HPMA, acrylate, methacrylate,acrylamide, or methacrylamide) as the monomer attached to the targetingligand. In some embodiments, the targeting ligand is attached or to adifferent type of monomer than the first anticancer agent. In otherwords, the monomer unit attached to the targeting ligand is differentthan the monomer unit attached to the first anticancer agent. Thetargeting ligand may be, for example, RGDfK, EPPT1, or folate.

In any embodiment, the first anticancer agent may be attached to thefirst polymer carrier through a linker. Likewise, where there aremultiple anticancer agents and/or targeting ligands attached to apolymer carrier, any or all of them may be attached through a linker. Insome embodiments having at least two anticancer agents attached to onepolymer carrier, both are attached by linkers.

Embodiments include compositions where the first polymer carrier hasbetween about 5% and about 99.7% of underivatized monomer units. Inother embodiments, the polymer carrier includes between about 25% andabout 98% of underivatized monomer units. In some embodiments, thepolymer carrier includes between about 50% and about 98% ofunderivatized monomer units. In some embodiments, the polymer carrierhas more than about 5%, more than about 10%, more than about 20%, morethan about 25%, more than about 40%, more than about 50%, more thanabout 70%, more than about 80%, or more than 90% of underivatizedmonomer units. In some embodiments, the polymer carrier has less thanabout 99.5%, less than about 99.0%, less than about 98.0%, or less thanabout 95% of underivatized monomer units. The underivatized monomerunits may be, for example, the first monomer unit (i.e. 2-CPMA, 3-CPMA,2-APMA, or 3-APMA), or another underivatized monomer unit.

Embodiments include polymers where the first polymer carrier has betweenabout 0.1 mol % and about 20.0 mol % of derivatized monomer unitsattached to an anticancer agent. In some embodiments, the first polymercarrier has more than about 0.1 mol %, more than about 0.2 mol %, morethan about 0.5 mol %, more than about 1.0 mol %, more than about 2.0 mol%, more than about 5.0 mol %, or more than about 7.0 mol % ofderivatized monomer units attached to an anticancer agent. In someembodiments the first polymer carrier has less than about 20 mol %, lessthan about 15 mol %, less than about 10 mol % or less than about 5.0 mol% of derivatized monomer units attached to an anticancer agent.

Embodiments include compositions where the first polymer carrier hasbetween about 0.1 mol % and about 94.8 mol % of derivatized monomerunits attached to a targeting ligand. In some embodiments, the firstpolymer carrier has more than about 0.1 mol %, more than about 0.5 mol%, more than about 1.0 mol %, more than about 2.0 mol %, more than about5 mol %, more than about 10 mol %, more than about 20 mol %, more thanabout 40 mol % or more than about 50 mol % of derivatized monomer unitsattached to a targeting ligand. In some embodiments, the first polymercarrier has less than about 94.8 mol %, less than about 90 mol %, lessthan about 80 mol %, less than about 70 mol %, less than about 50 mol %,less than about 40 mol % or less than about 20 mol % of derivatizedmonomer units attached to a targeting ligand.

Embodiments include polymer based therapeutic compounds comprising ananticancer agent and a polymer carrier, and optionally a linker moleculeand optionally a targeting ligand such as RGDfK, EPPT1 peptide orfolate, wherein the anticancer agent, the polymer carrier, linkermolecule, and/or targeting ligand, such as RGDfK, EPPT1 peptide orfolate are attached to one another via a covalent bond.

There are a number of different ways the anticancer agent, the polymercarrier, the optional linker molecule and the optional targeting ligandcan be attached to one another. In some embodiments, the anticanceragent, the polymer carrier, and optionally a linker molecule andoptionally a targeting ligand can be directly attached to one another.Example embodiments are described below and shown schematically in FIG.2A-2J. For example, the anticancer agent may be attached to the polymercarrier via a covalent bond, as shown in FIG. 2A.

In other embodiments, as shown in FIG. 2B, a linker molecule is directlyattached to the polymer carrier via a covalent bond, and the anticanceragent is directly attached to the linker molecule via a covalent bond.

In other embodiments, as shown in FIG. 2C, an anticancer agent isdirectly attached to the polymer carrier via a covalent bond, and atargeting ligand is directly attached to the polymer carrier via acovalent bond.

In other embodiments, as shown in FIG. 2D, a linker molecule is directlyattached to the polymer carrier via a covalent bond, and the anticanceragent is directly attached to the linker molecule, and a targetingligand is directly attached to the polymer carrier via a covalent bond.

In other embodiments, as shown in FIG. 2E, a linker molecule is directlyattached to the polymer carrier via a covalent bond, and a targetingligand is directly attached to the linker molecule, and an anticanceragent is directly attached to the polymer carrier via a covalent bond.

In other embodiments, as shown in FIG. 2F, a linker molecule is directlyattached to the polymer carrier via a covalent bond, and the anticanceragent is directly attached to the linker molecule, and a targetingligand is directly attached to a different linker molecule, which isdirectly attached to the polymer carrier via a covalent bond.

In other embodiments, there may be two or more anticancer agents ofdifferent types. For example, in any of the previous embodiments, twodifferent anticancer agents may be used. In embodiments having two ormore different anticancer agents, the anticancer agents may be attacheddirectly to the carrier or to a linker attached to the carrier. In someembodiments having two or more different anticancer agents, at leastone, but not all, of the anticancer agents are attached to a linker, andthe linker is directly attached to the carrier. In other embodimentshaving two or more different anticancer agents, all of the anticanceragents are attached to separate linkers, and the separate linkers areattached directly to the polymer carrier. An illustration, shown in FIG.2G of a compound having two anticancer agents, each separated from thecarrier by separate linkers is shown below. In all cases, the linkersare optional, and may be the same or different.

Embodiments, as shown in FIG. 2H, for example having two or moredifferent anticancer agents may also have a targetting ligand, which isoptionally separated from the polymer carrier by a linker. An example ofa compound having two anticancer agents with separate linkers, and atargeting ligand, separated by a linker is illustrated below. In allcases, the linkers are optional, and may be the same or different.

In all embodiments using more than one linker, the linkers may be thesame or different. For example, where an anticancer agent and atargeting ligand are both separated from the polymer carrier by alinker, the linkers may be the same, or different. Likewise, when two ormore anticancer agents are used, and more than one or all are separatedfrom the polymer carrier by linkers, the linkers may be the same, ordifferent.

Embodiments also include compositions and methods that use more than onepolymer carrier. For example, a composition may include a first polymercarrier having attached thereto an anticancer agent, optionally througha linker (e.g. FIG. 2A or 2B). The second polymer may also include atargeting ligand (e.g. FIG. 2C, 2D, 2E, or 2F). The composition can alsoinclude, for example, a second polymer carrier having attached thereto atargeting ligand, optionally through a linker. (e.g. FIG. 2I or 2J).

In these some embodiments, the second polymer carrier is not limited topolymers having 2-CPMA, 3-CPMA, 3-APMA, or 2-APMA monomers. For example,the second polymer carrier may be a polymer havingN-(2-hydroxypropyl)methacrylamide (HPMA),N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(3-aminopropyl)methacrylamide (3-APMA),N-(2-aminopropyl)methacrylamide (2-APMA) or other acrylate andacrylamide monomers. The monomers may be derivatized with an anticanceragent, a linker, a targeting ligand, a linker-anticancer agentcombination, or a linker-targeting ligand combination.

In some embodiments, the first and second polymer carriers may be linkedtogether to form a single larger polymer. In such embodiments, thepolymers may be linked through any linker capable of attaching to amonomer on each of the polymers. For example, in the first polymer, thelinking group may be attached to a monomer that is polymerized to formthe first carrier molecule, where the unattached terminus of the linkinggroup has a reactive functional group or leaving group that can bejoined to a reactive group on a monomer (or linker) on the secondcarrier polymer. For example, the first carrier polymer can include alinking group that terminates in a leaving group such as apara-nitrophenoxy (ONp) group. Such a carrier polymer can be reactedwith a second carrier molecule that includes an amine group, for examplewhere the second carrier molecule is prepared from 3-APMA. The twocarrier polymers can then be linked by displacement of the ONp group bythe amine group. In exemplary embodiments of a linked polymer, thelinking group is susceptible to cleavage by intracellular hydrolysis orby lysosomal enzymes to release the two separate polymer carriers.

Other compositions can include other combinations of the variousdelivery systems described above and illustrated in FIGS. 2A-2J. Methodsof treatment include coadministration of any of these polymer deliverysystem combinations.

The anticancer agent, the polymer carrier, the linker molecule and thetarget ligand used to produce the compounds are discussed below.

1. Anticancer Agent

An “anticancer agent” means any agent useful to combat cancer. Anyanticancer agent may be used that can be directly or indirectly attachedto the polymer carrier and/or the linker. A partial list of anticanceragents that can be used with the disclosed compositions can be found in,for example, U.S. Pat. No. 5,037,883, which is herein incorporated byreference as well as any publications and patents, or patentapplications, cited therein which contain anticancer agents. U.S. Pat.Nos. 6,348,209, 6,346,349, and 6,342,221 also describe agents related toanticancer compounds. Classes of anticancer agents include, but are notlimited to, chemotherapeutic agents, cytotoxins, antimetabolites,alkylating agents, protein kinase inhibitors, anthracyclines,antibiotics, antimitotic agents (e.g. antitubulin agents),corticosteroids, radiopharmaceuticals, and proteins (e.g. cytokines,enzymes, or interferons). Anticancer agents include, for example, smallmolecule organic compounds, macromolecules, metal containing compounds,and compounds or chelates that include radionuclides. In exampleembodiments, the anticancer compound is a small molecule organiccompound. Specific examples include, but are not limited to docetaxel,gemcitabine, imatinib (Gleevec®), 5-fluorouracil, 9-aminocamptothecin,amine-modified geldanamycin, doxorubicin, paclitaxel (Taxol®),cisplatin, procarbazine, hydroxyurea, meso e-chlorin, Gd(+3) compounds,asparaginase, and radionuclides (e.g 1-131, Y-90, In-111, and Tc-99m).There are many anticancer agents known in the art and many continue tobe developed. In some embodiments, the “anticancer agent” is docetaxelor gemcitabine. Some embodiments include two or more anticancer agents.Some embodiments include two anticancer agents. Some embodiments includedocetaxel and gemcitabine.

In some embodiments, the first anticancer agent is docetaxel,gemcitabine, cisplatin, derivatives of docetaxel, derivatives ofgemcitabine, or derivatives of cisplatin. In some embodiments having atleast a first and second anticancer agent, at least one of the first andsecond anticancer drug moieties is docetaxel, gemcitabine, cisplatin,derivatives of docetaxel, derivatives of gemcitabine, or derivatives ofcisplatin. In other embodiments' having a first and second anticanceragent, the first anticancer drug moiety is docetaxel or a derivativethereof and the second anticancer drug moiety is gemcitabine, cisplatinor a derivative thereof.

As will be appreciated, the anticancer agent, as attached to the polymercarrier, is modified compared to the anticancer agent per se. One ofordinary skill will be able to make the necessary chemical modificationsof the anticancer agent for attaching the anticancer agent to thepolymer carrier or linking molecule based on the description below.Making a chemical modification to attach an anticancer agent produces a“derivative” of that anticancer agent, as used herein. For example, a“derivative” of docetaxel, gemcitabine or cisplatin include chemicallymodified analogs of docetaxel, gemcitabine or cisplatin that enableattachment of docetaxel, gemcitabine, or cisplatin to the polymercarrier or linker. The derivative should not interfere with the activityof the anticancer agent. In embodiments having a cleavable linker, uponbond cleavage, the anticancer agent, or an active derivative thereof, isreleased.

2. Polymer Carrier

In one embodiment, the polymer carrier is a polymer produced by thepolymerization of an unsaturated monomer. Examples of monomers include,but are not limited to, acrylates and methacrylates, acrylamides, andmethacrylamides. In some embodiments of this invention, the polymercarrier is a homopolymer produced from the polymerization ofN-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(2-aminopropyl)methacrylamide (2-APMA),N-(3-aminopropyl)methacrylamide (3-APMA). In other embodiments, thepolymer carrier is a heteropolymer or copolymer produced from thepolymerization of two or more comonomer units where at least one monomerunit is N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(2-aminopropyl)methacrylamide (2-APMA),N-(3-aminopropyl)methacrylamide (3-APMA).

As used here, a polymer carrier that is a homopolymer of 2-CPMA, 3-CPMA,2-APMA, or 3-APMA means that the polymer backbone is derived from2-CPMA, 3-CPMA, 2-APMA or 3-APMA, but individual monomer units may beattached to, for example, a linker, an anticancer agent, a targetingligand, a linker attached to an anticancer agent, or a linker attachedto a targetting ligand.

A polymer carrier that is a heteropolymer or copolymer means that thepolymer backbone is produced from two or more comonomers where at leastone comonomer is 2-CPMA, 3-CPMA, 2-APMA, or 3-APMA, but the individualmonomer units may be attached to, for example, a linker, an anticanceragent, a targeting ligand, a linker attached to an anticancer agent, ora linker attached to a targetting ligand, as described above. In someembodiments, the 2-CPMA, 3-CPMA, 2-APMA or 3-APMA monomer may beattached to, for example a linker, an anticancer agent, a targetingligand, a linker attached to an anticancer agent, or a linker attachedto a targetting ligand. In some embodiments, a comonomer that is not2-CPMA, 3-CPMA, 2-APMA or 3-APMA may be attached to, for example, alinker, an anticancer agent, a targeting ligand, a linker attached to ananticancer agent, or a linker attached to a targetting ligand.

The carrier polymer molecule is a large macromolecule of at least 5,000daltons. The polymer carrier can range from about 5,000, to about1,000,000 daltons, from about 5,000 daltons to about 100,000 daltons,from about 5,000 to about 25,000 daltons, from about 25,000 to about100,000 daltons, from about 25,000 daltons to about 1,000,000 daltons,or from 100,000 daltons to 1,000,000 daltons. In embodiments where afirst and second polymer carrier are coupled or linked, the resultpolymer of at least 50,000 daltons, or at least about 100,000 daltons.For example, the resultant polymer can range from 50,000 daltons toabout 1,000,000 daltons, 100,000 daltons to about 1,000,000 daltons or100,000 daltons to about 250,000 daltons The polymer carrier aids in thetransport of an anticancer agent across the cell membrane. Thus, whenthe anticancer agent is directly or indirectly attached to the polymercarrier it typically crosses a cell membrane better than the anticanceragent alone. Some examples of polymer carriers are also described inU.S. Pat. No. 5,258,453 for “Drug delivery system for the simultaneousdelivery of drugs activatable by enzymes and light;” U.S. Pat. No.5,037,883 for “Synthetic polymeric drugs;” U.S. Pat. No. 4,074,039 for“Hydrophilic N,N-diethyl acrylamide copolymers;” U.S. Pat. No. 4,062,831for “Copolymers based on N-substituted acrylamides, N-substitutedmethacrylamides and N,N-disubstituted acrylamides and the method oftheir manufacturing;” U.S. Pat. No. 3,997,660 for “Soluble hydrophilicpolymers and process for producing the same;” U.S. Pat. No. 3,931,123for “Hydrophilic nitrite copolymers;” and U.S. Pat. No. 3,931,111 for“Soluble hydrophilic polymers and process for processing the same” eachof which is individually and specifically herein incorporated byreference in its entirety. These polymers can be modified according tothe present invention by incorporation of 2-CPMA, 3-CPMA, 2-APMA or3-APMA monomers. Alternatively, the polymers mentioned above can be usedin combination with polymers according to the present invention, forexample, as a second polymer carrier.

3. Linker Molecule

A “linker” refers to a group that spatially separates an anticanceragent or a targeting ligand from the polymeric backbone. The linker canbe any sort of entity, such as, without limitation, a poly (ethyleneglycol), an amino acid or a poly (amino acid), one end of which iscapable of forming a covalent bond with the polymer backbone and theother end of which is capable of forming a covalent bond with drug or atargeting ligand. The linkers may be cleavable so that the anticanceragent can be released, for example, under reducing conditions, oxidizingconditions, or by hydrolysis of an ester, amide, hydrazide, or similarlinkage that forms the covalent bond between the linker and theanticancer agent. Additionally, the type of linker may augment theselective cytotoxicity (and thus improve the therapeutic index) aspectby permitting selective release of the anticancer agent inside thecells. The structure of the linker may be tailor-made so as to be stablein the blood stream, yet susceptible to hydrolysis by lysosomal enzymesintracellularly. Oligopeptide sequences, oligosaccharide sequences orstructures similar to those in nucleic acids also may be used as pointsof drug attachment. The linkages or peptide spacers can be any of thosementioned in U.S. Pat. No. 5,037,883, for example, Gly-Gly, Gly-Phe-Gly,Gly-Phe-Phe, Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly-Leu-Phe,Gly-Leu-Ala, Ala-Val-Ala, Gly-Phe-Leu-Gly (SEQ ID NO: 1),Gly-Phe-Phe-Leu (SEQ ID NO: 2), Gly-Leu-Leu-Gly (SEQ ID NO: 3),Gly-Phe-Tyr-Ala (SEQ ID NO: 4), Gly-Phe-Gly-Phe (SEQ ID NO: 5),Ala-Gly-Val-Phe (SEQ ID NO: 6), Gly-Phe-Phe-Gly (SEQ ID NO: 7),Gly-Phe-Leu-Gly-Phe (SEQ ID NO: 8), or Gly-Gly-Phe-Leu-Gly-Phe (SEQ IDNO: 9). In some embodiments, the linker is a peptide spacer. In someembodiments, the linker is Gly-Phe-Leu-Gly (SEQ ID NO: 1). This spacerwill be repeatedly referred to throughout the specification and claimseither as Gly-Phe-Leu-Gly (SEQ ID NO: 1) or GFLG (SEQ ID NO: 1), whichterms can be used interchangeably. In some embodiments having at leastone linker, the linker is susceptible to cleavage by lysosomal enzymesand the linker can be, for example, oligopeptide sequences,oligosaccharide sequences or structures similar to those in nucleicacids. As used herein, a “structure similar to those in nucleic acids”means an oligonucleotide sequence having a phosphodiester-riboselinkage. These linkages may be cleaved, for example, byphosphodiesterases, DNAses, RNAses, and endonucleases. Examples ofenzymes that cleave oligopeptides include proteases and peptidases.Examples of enzymes that cleave oligosaccharides include sugarhydrolases and glycosidases

4. Targeting Ligand

The term “targeting ligand” means a molecule which serves to deliver thecompound of the invention to a specific site for the desired activity.Targeting ligands include, for example, molecules that specifically bindmolecules on a specific cell surface. Example targeting ligands includeantibodies, antibody fragments, small organic molecules, peptides,peptoids, proteins, polypeptides, oligosaccharides, transferrin,HS-glycoprotein, coagulation factors, serum proteins, beta-glycoprotein,G-CSF, GM-CSF, M-CSF, EPO, and the like. In some embodiments, thetargeting ligand is RGDfK, EPPT1 peptide, or folate, which is attachedto the polymer, optionally via a linker.

Generally with passively targeted HPMA conjugates success in clinicaltrials has been marginal primarily because of the limited accumulationof the drug in solid tumors by passive diffusion alone and heterogeneityof clinical presenting cancers. Active targeting strategies may allowtargeting to multiple cell types taking into account the variations intumor physiology, maximize distribution in the microenvironment of solidtumors while concurrently minimizing their non-specific uptake in otherorgans. Active targeting strategies will also significantly improve thetherapeutic efficacy by (1) increasing tumor specificity; (2) improvingpharmacokinetics; and (3) reducing toxicity. Several such strategieshave emerged over the recent years that can be exploited tosignificantly improve tumor localization of anticancer drugs. Activetargeting of polymeric drug delivery systems by attaching molecularmarkers (e.g., peptides and antibodies) has shown to significantlyimprove tumor localization.

Mucin-1 is a transmembrane molecule, expressed by most glandularepithelial cells. Several important features make mucin-1 an attractivereceptor for targeted delivery to tumors.

First, mucin-1 is overexpressed on almost all human epithelial celladenocarcinomas, including 90% of human breast, ovarian, pancreatic,colorectal, lung, prostate, colon, and gastric carcinomas. Moreover,mucin-1 expression has been demonstrated in nonepithelial cancer celllines (astrocytoma, melanoma, and neuroblastoma), as well as inhematological malignancies such as multiple myeloma and some B-cellnon-Hodgkin lymphomas, in total constituting 50% of all cancers inhumans.

Second, in adenocarcinomatous tissue, as the result of the lost glandarchitecture, mucin-1 is ubiquitously expressed all over the cellsurface. Because of its rod-like structure, the molecule extends 100-200nm above the surface, which is 5-10-fold the length of most membranemolecules. This feature makes mucin-1 an accessible target fortherapeutic probes.

Third, whereas in normal tissues mucin-1 is heavily glycosylated (50-90%of its molecular mass is due to carbohydrates), mucin-1 is typicallyunderglycosylated in neoplastic tissues. Reduced glycosylation permitsthe immune system to access the peptide core of the tumor-associatedunderglycosylated mucin-1 antigen and reveals epitopes, which in thenormal cell are masked. This feature makes it possible to design probesthat discriminate between normal cells and adenocarcinoma cells.

Fourth, the extracellular domain of mucin-1, defined by the presence ofthe APDTRP (SEQ ID NO: 10) sequence, extends above the cell surface,thus interfering with the interaction between adhesion molecules on thetumor cell surface and their ligands on lymphocytes, aiding in theinaccessibility of tumor epitopes to immune recognition. Therefore,there is no tendency for tumor antigen down-regulation in response toimmunotherapy, and mucin-1 expression remains homogeneously up-regulatedduring the life of the tumor and tumor metastases. These features areimportant in designing targeted drug delivery for different stages oftumor progression.

An abundant number of investigations have focused on the potential touse mucin-1 as a target for immunotherapy. Multiple monoclonalantibodies have been produced to recognize the immunogenic APDTRP (SEQID NO: 10) sequence of the tandem repeat. However, when antibodies wereused as targeting molecules, the immunogenicity and long plasmahalf-life of these proteins were detrimental. Consequently, the use ofsmall peptides instead may eliminate these shortcomings because peptideligands are nonimmunogenic and have high affinity and selectivity forreceptors. Synthetic peptide, designated EPPT1 (YCAREPPTRTFAYWG; SEQ IDNO: 11), has been developed as specific ligands and has shownsignificant affinity (Kd=20 μM). EPPT1 peptide, labeled with (99 mTc),has been used to image breast carcinomas in vivo. All of the features ofthe mucin-1 protein listed above make this molecule an ideal candidatefor a potential tumor targeting ligand.

A number of tumor cell and associated vasculature specific receptorshave also been identified that differentiate tumor cells from normalcells. The αVβ3 integrin is one of the most studied and is selectivelyoverexpressed in tumor associated neovasculature as well as in certainmetastatic cancers (Felding-Habermann et al., Clin. Exp. Metastasis, 19:427-436 (2002)). High affinity αVβ3 selective ligands containing thetripeptide sequence, Arg-Gly-Asp (RGD), have been identified by phagedisplay studies. The conformationally restrained RGD sequence, i.e.cyclic RGD, contains disulfide bridges and binds to αvβ3 20-40 fold moreavidly than linear RGD peptides (Koivunen, E., Wang, B. & Ruoslahti, E.,Biotechnology (N.Y.), 13: 265-270 (1995)). RGD peptide has beenconjugated with doxorubicin (Arap, W., Pasqualini, R. & Ruoslahti, E.,Science, 279: 377-380 (1998)) for targeted chemotherapy as well as fortargeted radiotherapy (Capello, A. et al., J. Nucl. Med., 45: 1716-1720(2004)). They have been conjugated to humanized antibodies, liposomes,poly (ethylene glycol) and HPMA copolymers to improve biodistributionand increase tumor accumulation and antitumor efficacy. These studiesmake RGD and cyclic RGD an ideal targeting ligand for studyinganti-tumor drug targeting.

Folic acid and its reduced counterparts are required by eukaryotic cellsfor one carbon transferreactions used in the biosynthesis of nucleotidebases. Cellular uptake of folates is facilitated by either a lowaffinity reduced folate carrier (Km˜1 μM), which is present in virtuallyall cells of the body, or a high affinityglycosylphosphatidylinositol-linked folate receptor (FR) (KD=˜100 pM),which exhibits highly limited distribution. FRs exhibit limitedexpression on healthy cells, but are often present in large numbers oncancer cells. For example, FRs are overexpressed on epithelial cancersof the ovary, mammary gland, colon, lung, prostate, nose, throat, andbrain. FRs are also overexpressed on hematopoietic malignancies ofmyeloid origin, including chronic and acute myelogenous leukemias. Astrong correlation has been observed between FR expression and the gradeand histological stage of a tumor. A variety of folate linked moleculesand complexes have been designed to enable selective delivery of drugsto FRs on cancer cells and activated macrophages. Other features thatrender folic acid an attractive ligand for use in drug targeting includeits low molecular weight (MW 441), water solubility, stability todiverse solvents, pHs, and heat, facile conjugation chemistry, lack ofimmunogenicity, and high affinity for its receptor.

In some embodiments that include a targeting ligand, the targetingligand may be RGDfK, EPPT1, or folate. In some embodiments, thetargeting ligand is attached to the carrier polymer by a linker, whichis an amino acid or peptide. In some embodiments, the linker is apeptide comprising Gly-Gly.

5. Efficiency and Specificity of Uptake by the Cells

The polymer based therapeutic compounds described herein can becharacterized in that they allow for the uptake of anticancer agents bycells using typically different mechanisms than used by the anticanceragent alone. There are many ways to determine whether the efficiencyand/or specificity of the uptake are increased by the polymer carrier.Typical increases of efficiency and/or specificity can be greater thanor equal to at least 2 fold, 5 fold, 10 fold, 25 fold, 50 fold, 100fold, 500 fold, 1000 fold, 5,000 fold or 10,000 fold.

6. Combination of Two Anticancer Agents

In some embodiments, the polymer based therapeutic composition haseither a single copolymer having two (or more) anticancer agentsattached thereto or a mixture of two or more copolymers, one containinga first anticancer agent and the other containing a second differentanticancer agent. In some embodiments, the two anticancer agents differin mechanism of action, or anticancer effect, etc. The anticancer agentscan be bound to the polymeric carrier(s) via bonds stable in the bloodstream, but susceptible to cleavage by lysosomal enzymes. When soformulated, both anticancer agents enter the same cells almostconcurrently because the body distribution of both anticancer agentswill generally be the same. This is fundamentally different compared tothe combination therapy of two low molecular weight drugs not attachedto polymer chains because the body distribution of each drug can bedifferent if the drugs are not each attached to a polymer carrier.Moreover, after reaching the lysosomal compartment of the cell, theanticancer agents bound via an enzymatically degradable bond arereleased from the carrier by the action of lysosomal enzymes and diffusethrough the lysosomal membrane into the cytoplasm. One of the mainadvantages of this approach is the optimization of the action of bothanticancer agents by using anticancer agents that have differentmechanism of action in cancer treatment. This will cause the death ofcancer cells which were not destroyed by one anticancer drug.

The combination effect of anticancer agents may be obtained byadministration of two separate copolymers, one containing an anticanceragent and the other containing another anticancer agent, whenadministered at the same time, when compared to the administration ofeach polymer administered separately in treating neoplastic diseases.Also, a single copolymer containing two different anticancer agents canbe utilized instead of a mixture of copolymers. The specificity of thesecopolymers may be improved by the attachment of a targeting ligand toeach polymer molecule.

Polymeric macromolecules typically are believed to enter targeted cellsby pinocytosis; binding low molecular weight anticancer agents tocopolymers alters their manner of uptake from diffusion to pinocytosiswhich may reduce the side effects normally elicited by the freeanticancer agents. For this reason it is possible to use much lowerdoses of both anticancer agents when attached to the “combination”copolymer. In addition, it is possible to use even lower doses if thetwo anticancer agents have a synergistic anticancer effect. Attachingboth anticancer agents to the same copolymer ensures that bothanticancer agents will enter the same cell at the same time. A targetingligand specific for a tumor marker on the cancer cell also bound to the“combination” copolymer side chains will facilitate or enhance thedirection of the copolymer containing both anticancer agentsspecifically to the targeted cancer cells.

In U.S. Pat. No. 5,258,453, the antitumor efficacy of the combinationcopolymers (such as HPMA copolymers), containing an anticancer drug(such as adriamycin) and containing a photosensitizer (such asmeso-chlorin e6 monoethylene diamine disodium salt (ce₆)) in vivo wasfound to be superior to the use of copolymers containing thephotosensitizer and polymers containing the anti-cancer drugadministered alone.

The present invention can also minimize the amount of cancer cells whichare resistant to chemotherapy, thus decreasing substantially thepossibility of tumor recurrence. This approach can be more successful inthe treatment of multidrug resistant cells (MDR) than presentlyavailable therapies. The concentration of anticancer agents in the cell,when this method is used, is increased, even if the transport of theanticancer agents into the cell interior or MDR cells is impaired. If asuitable targeting ligand is attached (e.g. structures complementary tocell surface antigens or receptors), then a combined intracellular andextracellular action will increase the efficacy (the intracellularaction will proceed by the above described mechanism, the extracellularaction will be on the plasma membrane).

C. Method of Making Compounds

The present invention includes the synthesis of N-2-carboxypropylmethacrylamide (2-CPMA), N-(3-carboxypropyl)methacrylamide (3-CPMA), andN-(2-aminopropyl)methacrylamide (2-APMA).

In most instances, the main comonomer unit determines the properties ofthe polymeric carriers. Several comonomer units may be used resulting inwater soluble copolymers. The copolymer can be made by copolymerizationof the desired mole ratio of underivatized comonomer units with thedesired ratio of comonomer units which have been derivatized to containappropriate attachment groupings or spacers or linkers, which, in turn,possess reactive groups to which bioactive agents, such as anticanceragents, or targeting moieties may be subsequently attached. Inalternative embodiments, the use of comonomers with different functionalgroups allows for selectively derivatizing one comonomer. Thus, thepolymer can also be derivatized after polymerization. Typical comonomerunits may be made of N-(2-hydroxypropyl)methacrylamide (HPMA),N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(2-aminopropyl)methacrylamide (2-APMA) orN-(3-aminopropyl)methacrylamide (3-APMA). Other suitable carriersinclude polyamino acids, polysaccharides, copolymers containingpolyethyleneoxide sequences, polyvinyl pyrrolidone-maleic anhydridecopolymers, and the like.

The comonomers utilized in this invention includeN-(2-hydroxypropyl)methacrylamide (HPMA),N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),(N-(2-aminopropyl)methacrylamide (2-APMA)) orN-(3-aminopropyl)methacrylamide (3-APMA) (available from Polysciences,Inc (PA)).

The basic comonomer, N-(2-aminopropyl)methacrylamide (2-APMA), can bemade, for example by the following process.

Embodiments include the acidic drug carrier, such as 2-CPMA or 3-CPMApolymers and copolymers, or other drug carriers such as neutral HPMApolymers and copolymers, and basic drug carrier such as 3-APMA or 2-APMApolymers and copolymers, where the polymer or copolymer includes atleast one of 2-CPMA, 3-CPMA, 2-APMA or 3-APMA monomers, as usefulpolymeric systems for the delivery of one or two anticancer agents.

Using the monomer units, N-(2-hydroxypropyl)methacrylamide (HPMA),N-(2-carboxypropyl)methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(3-aminopropyl)methacrylamide (3-APMA), orN-(2-aminopropyl)methacrylamide (2-APMA), polymers and copolymers can beprepared and used as carriers for the delivery of anticancer agents tothe cancer cell. Copolymers containing two or more of HPMA, 2-CPMA,3-CPMA, 2-APMA and 3-APMA can also be prepared in similar manner.

The compositions of the invention can be prepared using techniques knownin the art. As described, there are up to four components used toproduce the compositions: the anticancer agent, the polymer carrier, thetargeting moiety and a linker molecule. Any of the components can bereacted with one another in any suitable order or combination to producethe compounds of the invention. It is sometimes preferred to couple(i.e., react) two of the components together to produce a new reactionproduct or intermediate, and then chemically connect the intermediatewith the next component. Polymerization can be performed either beforeor after derivatization of a monomer with a linker, anticancer agent,and/or targeting ligand.

For example, the anticancer agent can be reacted with a monomer, (forexample, 2-CPMA, 3-CPMA, 2-APMA, 3-APMA or HPMA) to produce aderivatized monomer, in this case an anticancer/monomer molecule. Theanticancer/monomer molecule can then be polymerized with the same orother monomers to produce a composition according to the invention.Alternatively, the anticancer agent can be reacted with a linkermolecule to produce an anticancer/linker molecule, which can then bereacted with a monomer to produce a derivatized monomer, in this case aan anticancer/linker/monomer molecule. The anticancer/linker/monomermolecule can then be polymerized with the same or other monomers toproduce a composition according to the invention. As yet anotheralternative, one or more monomers can be polymerized and the resultantpolymer reacted with an anticancer agent or linker/anticancer agentmolecule to form a composition according to the invention. Targetingligands (optionally through a linker) can be incorporated into thecomposition in a similar manner by preparing monomer molecules followedby polymerization or by reaction with a preformed polymer.

As is apparent, there are a number of combinations and ordering of stepsthat can be used to produce carrier polymer compositions according tothe invention. In embodiments where the composition includes more thanone carrier polymers, each polymer can be separately produced and thencombined to generate the composition.

For example, in one embodiment, the compound can be produced by (1)reacting the linker with a monomer to produce a monomer/linker molecule,(2) reacting the monomer/linker molecule with an anticancer agent and(3) polymerizing the monomers (with any comonomers), followed by addinga targeting ligand.

In a specific example, the polymer-based therapeutic compound can beproduced by reacting methacryloyl chloride (MACl) with Gly-Phe (GF) andcoupling the product with Leu-Gly (LG) to produce an MA-GFLG-OH molecule(see Scheme 3 below). The MA-GFLG-OH molecule is then reacted withgemcitabine or docetaxel to produce the MA-GFLG-anticancer molecule(s).The MA-GFLG-anticancer molecule(s) is then reacted (polymerized) with atleast one of 2-CPMA, 3-CPMA, 2-APMA, or 3-APMA comonomer, and optionallyother comonomers to produce a polymer-based therapeutic compound in theform of a copolymer-drug(s) conjugate. The copolymer-drug(s) conjugatemay then be reacted with a targeting ligand, such as, for example,RGDfK, EPPT1 peptide or folate a polymer-based therapeutic compoundcontaining a targeting ligand. ‘GFLG’ is disclosed as SEQ ID NO: 1

In another embodiment, MA-GFLG-OH is reacted with Docetexal (DCT) togive MA-GFLG-DCT and MA-GFLG-ONp with Gemcitabine (GEM) to giveMA-GFLG-GEM. MA-GFLG-ONp is the p-nitrophenyl ester of MA-GFLG-OH. Thesetwo monomers are reacted (or polymerized) with 2-CPMA, 3-CPMA, 2-APMA,and/or 3-APMA comonomer to produce a copolymer. Additional comonomersmay be included in the reaction, such as, for example, HPMA and/ormethacryloyl-glycylglycine-O-p-nitrophenylester (MA-GG-ONp) to produceother copolymers. A copolymer produced from a reaction includingMA-GG-ONp can be further reacted with a targeting ligand to produce apolymer-based therapeutic compound. HPMA or 2-CPMA or 3-CPMA or 2-APMAor 3-APMA-GFLG-drug containing a targeting ligand, including, forexample, RGDfK, EPPT1 peptide or folate. ‘GFLG’ is disclosed as SEQ IDNO: 1

As described above, the anticancer agent, polymer carrier, and linkercan be attached to one another directly or indirectly. In addition, theattachment of each component to one another can vary depending upon thetypes of components selected and the order in which the components arepermitted to react with one another.

D. Method of Using Compounds

The disclosed polymer-based therapeutic compounds can be used forpassive or targeted delivery of anticancer agents to cells. Thesecompounds can be used to treat a variety of disorders that require thedelivery of anticancer or similar agents. It is understood that any ofthe compounds disclosed can be used in this way. Those of skill in theart understand the compounds will be administered in pharmaceuticallyacceptable forms (i.e. in a pharmaceutical composition) and in doseswherein delivery occurs. Typically the compounds would be administeredto patients in need of delivery of the anticancer agent or a similarcompound. It is understood that the goal is delivery of the compound tothe cells of the patient in need of the anticancer agent or similaragent.

The polymer based therapeutic compounds, bearing conjugated anticanceragents, can be given to a subject. Any subject in need of receiving ananticancer agent can be given the disclosed conjugated anticanceragents. The subject can, for example, be a mammal, such as a mouse, rat,rabbit hamster, dog, cat, pig, cow, sheep, goat, horse, or primate, suchas monkey, gorilla, orangutan, chimpanzee, or human.

The polymer based therapeutic compounds, bearing conjugated anticanceragents, can be used for inhibiting cancer cell proliferation. Inhibitingcancer cell proliferation means reducing or preventing cancer cellgrowth. Inhibitors can be determined by using a cancer cell assay. Forexample, either a cancer cell line can be cultured on 96-well plates inthe presence or absence of the conjugated anticancer agent or anticanceragent alone or anticancer agent prepared differently then the disclosedcompositions (for example, just anticancer agent and carrier) for anyset period of time. The cells can then be assayed. In certainembodiments the conjugated anticancer compounds are those that willinhibit 10% or 15% or 20% or 25% or 30% or 35% or 40% or 45% or 50% or55% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% of growthrelative to any of the controls as determined by the assay.

Disclosed are compositions which inhibit metastatic tumor formation inthis type of assay disclosed herein, as well as compositions that reducemetastatic tumor formation by at least 10% or 15% or 20% or 25% or 30%or 35% or 40% or 45% or 50% or 55% or 60% or 65% or 70% or 75% or 80% or85% or 90% or 95% of a control compound.

The disclosed compositions can be used to treat any disease whereuncontrolled cellular proliferation occurs such as cancers or neoplasticdisorders. A non-limiting list of different types of cancers is asfollows: carcinomas, carcinomas of solid tissues, squamous cellcarcinomas, adenocarcinomas, sarcomas, gliomas, high grade gliomas,blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas,adenomas, hypoxic tumours, myelomas, metastatic cancers, or cancers ingeneral.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, and epithelialcancer, renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,or pancreatic cancer.

Compositions disclosed herein may also be used for the treatment ofprecancer conditions such as cervical and anal dysplasias, otherdysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, andneoplasias.

E. Dosages

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which delivery occurs. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any counterindications. Dosage can vary from about 1 mg/kg to30 mg/kg in one or more dose administrations daily, for one or severaldays.

F. Pharmaceutically Acceptable Carriers

Any of the compositions can be used therapeutically in combination witha pharmaceutically acceptable carrier to form a pharmaceuticalcomposition.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration ofcompositions to humans, including solutions such as sterile water,saline, and buffered solutions at physiological pH. Other compounds willbe administered according to standard procedures used by those skilledin the art.

Compositions intended for pharmaceutical delivery may be formulated in apharmaceutical composition. Pharmaceutical compositions may includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions may also include one or more activeingredients such as antimicrobial agents, anti-inflammatory agents,anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions that may also containbuffers, diluents and other suitable additives. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.

The compositions as described herein can also be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Pharmaceutical Compositions

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The dosage regimen for treating a disease condition with the compoundsand/or compositions of this invention is selected in accordance with avariety of factors, including the type, age, weight, sex, diet andmedical condition of the patient, the severity of the disease, the routeof administration, pharmacological considerations such as the activity,efficacy, pharmacokinetic and toxicology profiles of the particularcompound employed, whether a drug delivery system is utilized andwhether the compound is administered as part of a drug combination.Thus, the dosage regimen actually employed may vary widely and thereforemay deviate from the preferred dosage regimen set forth above.

Injectable preparations, including, for example, sterile injectableaqueous or oleaginous suspensions may be formulated according to theknown art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

While the compositions of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more therapeutic agents, such as immunomodulators, antiviralagents or antiinfective agents.

The foregoing is merely illustrative of the invention and is notintended to limit the invention to the disclosed compositions.Variations and changes that are obvious to one skilled in the art areintended to be within the scope and nature of the invention which aredefined in the appended claims. From the foregoing description, oneskilled in the art can easily ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

EXAMPLES

The invention may be further clarified by references to the followingexamples, which serve to exemplify some of the preferred embodiments,and not to limit the invention in any way.

Example 1 Synthesis of Polymer-Gemcitabine or Docetaxel orGemcitabine/Docetaxel Conjugates 1) Synthesis ofN-(2-carboxypropyl)methacrylamide (2-CPMA) Comonomers

Synthesis of the reactive monomer 2-CPMA was described as shown inScheme 1.

3-Aminoisobutyric acid (5.5 g, 53.3 mmol) was dissolved in 26.8 ml of 2NNaOH (53.6 mmol) and cooled to −5° C. Freshly distilled methacryloylchloride (MACl) (7.9 g, 75.9 mmol) in 22 ml of dichloromethane was addeddropwise. A small amount of inhibitor, tertiary octyl pyrocatechine wasadded to prevent polymerization of the monomer. Simultaneously but witha slight delay, 38.5 ml (76.9 mmol) of 2N NaOH was added dropwise to thereaction mixture. After addition of MACl and NaOH the reaction mixturewas warmed up to room temperature and allowed to react for two hours.The pH was maintained at around 8-9. The dichloromethane layer wasseparated from the water layer, washed with water (20 ml×2) anddiscarded. The combined aqueous layer was mixed with 100 ml of ethylacetate. Under vigorous stirring and cooling, dilute HCl was addedslowly until the pH reached at 2. The organic layer was separated andthe aqueous layer was extracted three times with ethyl acetate. Thecombined organic layer was dried over anhydrous sodium sulfateovernight. The dried solution was filtered and washed with ethylacetate. The ethyl acetate was removed by rotary evaporation to obtainthe product as a white powder. Recrystallization was done from ethylacetate (8.5 g, 93.2% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.25 (s, 3H),1.96 (s, 3H)), 2.79-2.80 (m, 1H), 3.35-3.41 (m, 1H), 3.59-3.65 (m, H),5.34 (s, 1H), 5.71 (s, 1H), 6.44 (s, 1H, NH).

N-(3-carboxypropyl)methacrylamide (3-CPMA) can be made as above,synthesis of 2-CPMA, using methacryloyl chloride and 4-aminobutyricacid.

2) Synthesis of N-(2-hydroxypropyl)methacrylamide (HPMA) Comonomers

Synthesis of the reactive monomer HPMA was as previously described(Kopecek and Bazilova, 1973) as shown in Scheme 2.

To a solution of 1-amino-2-propanol (128.4 g, 1.71 mol) in 500 ml ofacetonitrile, freshly distilled methacryloyl chloride (MACl) (90.8, 0.86mol) in 40 ml of acetonitrile was added drop wise under vigorousstirring at −5° C. A small amount of inhibitor, tertiary octylpyrocatechine was added to the solution. The reaction mixture wasstirred for an additional 30 min at room temperature. 1-Amino-2-propanolhydrochloride formed as a byproduct was precipitated and filtered off.The residue was washed with pre-cooled acetonitrile. The filtrate wascooled to −70° C. and the HPMA precipitated. After equilibrating to roomtemperature the product was filtered off and washed with pre-cooledacetonitrile. Recrystallization was from acetone and the pure productwas isolated (75.9 g, 61.6% yield). MS (ESI) m/z 144 (M+1). ¹H NMR (400MHz, CDCl₃): δ 1.20 and 1.22 (d, J=6.4 Hz, 3H)), 1.97 (s, 3H), 3.18-3.21(m, 1H), 3.48-3.51 (m, 1H), 3.95-3.96 (m, 1H), 5.36 (s, 1H), 5.74 (s,1H).

3) Synthesis of MA-GF-OH

Methacryloylglycylphenylalanine (MA-GF-OH) was made from the reaction ofmethacryloyl chloride (MACl) and glycylphenylalanine (GF) as outlined inScheme 3.

Glycylphenylalanine (Gly-Phe), 5.0 g, 22.5 mmol) was dissolved in 11.3ml of 2N NaOH (22.5 mmol) and cooled to 0-5° C. Freshly distilled MACl(2.8 g, 26.8 mmol) in 10 ml of dichloromethane was added dropwise. Asmall amount of inhibitor, tertiary octyl pyrocatechine was added toprevent polymerization of the monomer. Simultaneously but with a slightdelay, 13.5 ml (26.9 mmol) of 2N NaOH was added dropwise to the reactionmixture. After addition of MACl and NaOH the reaction mixture was warmedup to room temperature and allowed to react for two hours. The pH wasmaintained at around 8-9. The dichloromethane layer was separated fromthe water layer, washed with water (7 ml×2) and discarded. The combinedaqueous layer was mixed with 50 ml of ethyl acetate. Under vigorousstirring and cooling, dilute HCl was added slowly until the pH reachedat 2-3. The organic layer was separated and the aqueous layer wasextracted three times with ethyl acetate. The combined organic layer wasdried over anhydrous sodium sulfate overnight. The dried solution wasfiltered and washed with ethyl acetate. The ethyl acetate was removed byrotary evaporation to obtain the product as a white powder.Recrystallization was done from ethyl acetate (6.3 g, 96.5% yield,melting point: 141.8-143.4° C.). ¹H NMR (400 MHz, CDCl₃): δ 1.96 (s,3H)), 3.06-3.20 (2m, 2H), 3.85-4.11 (2m, 2H), 4.83-4.85 (m, 1H), 5.41(s, 1H), 5.79 (s, 1H), 7.20-7.30 (m, 5H).

4) Synthesis of LG-OMe HCl

Leucylglycine-OMe (LG-OMe) was made from the reaction of leucylglycine(LG) and methanol/thionyl chloride as outlined in Scheme 3.

Leucylglycine (Leu-Gly, 5.0 g, 26.6 mmol) was dissolved in 40 ml ofmethanol and cooled to −15° C. An excess of thionyl chloride (SOCl₂) (4ml, 54.8 mmol) was added dropwise for 15 min under stirring. Afterequilibrating to room temperature the mixture was refluxed for 3.5hours. The solvent was evaporated to dryness and the residue wasdissolved in methanol and evaporated again to remove traces of HCl andSOCl₂. The residue was mixed with ethyl ether and the removal of ethylether layer and evaporation gave a white amorphous solid. The crudeproduct (LG-OMe.HCl, 6.53 g) was used in subsequent steps withoutpurification. ¹H NMR (400 MHz, DMSO-d₆): δ 0.89-0.93 (m, 6H), 1.56-1.61(m, 2H)), 1.71-1.78 (m, 1H), 3.65 (s, 3H), 3.77-3.85 (m, 2H), 3.88-4.00(m, 1H), 5.41 (s, 1H), 5.79 (s, 1H), 7.25-7.28 (m, 5H).

5) Synthesis of MA-GFLG-OMe (SEQ ID NO: 1)

Methacryloylglycylphenylalanylleucylglycine OMe (MA-GFLG-OMe) was madefrom the reaction of methacryloylglycylphenylalanine (MA-GF-OH) andleucylglycine-OMe (LG-OMe) as outlined in Scheme 3. ‘GFLG’ is disclosedas SEQ ID NO: 1.

5.1 g of crude Leu-Gly-OMe HCl (21.5 mmol) in 3.88 g ofN,N′-diisopropylethylamine (DIPEA, 30 mmol) and 50 ml of ethyl acetatewas mixed with 5.0 g of MA-Gly-Phe (17.2 mmol) in 200 ml of ethylacetate under stirring at room temperature. To the mixture, 18.3 g of(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate(BOP, 41.3 mmol) was added and the reaction mixture was stirred for 3days at room temperature. The reaction mixture was washed with 5% NaHCO₃solution (200 ml×3), water, 1M NaHSO₄ (200 ml×3) and saline. The organiclayer was dried over anhydrous sodium sulfate. After filtering off thedrying agent and the filtrate was concentrated under vacuum to obtainthe product (MA-GFLG-OMe). The purification by SiO₂ columnchromatography gave 6.35 g of MA-GFLG-OMe (77.8% yield, melting point:140.9-143.0° C.). ‘GFLG’ is disclosed as SEQ ID NO: 1.

6) Synthesis of MA-GFLG-OH (SEQ ID NO: 1)

Methacryloylglycylphenylalanylleucylglycine (MA-GFLG-OH) (SEQ ID NO: 1)was made from the hydrolysis ofmethacryloylglycylphenylalanylleucylglycine OMe (MA-GFLG-OMe) (SEQ IDNO: 1) as outlined in Scheme 3.

To a cooled solution of 5.1 g of MA-GFLG-OMe (SEQ ID NO: 1) (10.7 mmol)in 109 ml of methanol, excess of 1N NaOH (12.9 ml, 12.9 mmol) was addeddropwise under stirring at 0° C. After addition of a small amount ofinhibitor (t-octyl pyrocatechine) the reaction mixture was stirred fortwo hours at 0° C. and then for four hours at room temperature. Thereaction mixture was concentrated under vacuum to remove methanol, mixedwith 150 ml of distilled water. The water layer was washed with ethylacetate (100 ml×2) and acidified with 1.0 M citric acid to pH 2.0˜2.5.The free acid was extracted with 3×150 ml of ethyl acetate, washed withsaturated brine and dried over anhydrous sodium sulfate overnight. Afterevaporation of the solvent under vacuum the tetrapeptide product(MA-GFLG-OH) (SEQ ID NO: 1) was recrystallized from ethylalcohol/n-hexane (1:1) mixture (3.06 g, melting point: 161.4-165.6° C.).MS (ESI) m/z 483 (M+Na).

7) Synthesis of methacryloylglycylphenylalanylleucylglycyl-Gemcitabine(MA-GFLG-Gemcitabine (SEQ ID NO: 1)) (Scheme 4)

730 mg of MA-GFLG-OH (SEQ ID NO: 1) (1.52 mmol), 414 mg of gemcitabineHCl (1.38 mmol), 732 mg of BOP (1.66 mmol) and a small amount oftertiary octyl pyrocatechine were dissolved in the mixture of 0.5 ml ofDIPEA and 30 ml of acetonitrile under nitrogen at room temperature. Themixture was stirred for 1 day at room temperature and concentrated undervacuum to remove the solvent. The product was purified by columnchromatography (silica gel, eluent: EtOAc/MeOH=4/1) and analyzed by massspectrometry (M+1=706.3) and tlc. The yield was 962 mg (98.8%).

8) Synthesis of methacryloylglycylphenylalanylleucylglycyl-Docetaxel(MA-GFLG-Docetaxel (SEQ ID NO: 1)) (Scheme 5)

363 mg of MA-GFLG-OH (SEQ ID NO: 1) (0.76 mmol), 555 mg of docetaxel(0.69 mmol), 574 mg of BOP (1.30 mmol), 114 mg of4-dimethylaminopyridine (DMAP, 0.94 mmol) and a small amount of tertiaryoctyl pyrocatechine were dissolved in the mixture of 10 ml of ethylacetate and 12 ml of acetonitrile under nitrogen at 4° C. The reactionmixture was stirred for 1 h at 4° C. and then for 2 days at roomtemperature and concentrated under vacuum to remove the solvent. Theproduct was purified by column chromatography (silica gel, eluent:EtOAc/MeOH=10/1) to give 803 mg of the product (91.5% yield). Theproduct was verified by thin layer chromatography and mass spectroscopym/z 1272.3 (M+Na).

9) Polymer-Gly-Phe-Leu-Gly-Gemcitabine (SEQ ID NO: 1) Preparation

HPMA, 2-APMA, 3-APMA 2-CPMA or 3-CPMA copolymer-gemcitabine conjugateare synthesized from the comonomers, by free radical precipitationcopolymerization of the comonomers HPMA, 2-APMA, 3-APMA 2-CPMA or 3-CPMA(1.00 mmol) and MA-GFLG-gemcitabine (SEQ ID NO: 1) (78.4 mg, 0.112 mmol)in acetone (2 ml) at 50° C. for 24 h using N,N′-azobisisobutyronitrile(AIBN, 10.8 mg) as the initiator as shown in Scheme 6. Typically theratio of comonomers:initiator:solvent is kept constant at 12.5:0.6:86.9wt %. The mixture is sealed under nitrogen in an ampoule and left topolymerize with stirring at 50° C. for 24 h. The precipitated polymer isdissolved in methanol, reprecipitated in 20× volume of ether and washedwith ether. Small molecular weight unreacted monomers and otherimpurities are separated from the polymeric conjugates by redissolvingin distilled water and dialyzing against distilled water to remove thesalts and subsequently lyophilizing to obtain the pure product.

10) Polymer-Gly-Phe-Leu-Gly-Docetaxel (SEQ ID NO: 1) Preparation

HPMA, 3-APMA or 2-CPMA copolymer-docetaxel conjugate were synthesizedfrom the comonomers, by free radical precipitation copolymerization ofthe comonomers HPMA (7.13 mmol), 3-APMA (5.93 mmol) or 2-CPMA (6.15mmol) and MA-GFLG-docetaxel (SEQ ID NO: 1) (0.18 mmol for HPMA, 0.15mmol for 3-APMA and 0.16 mol for 2-CPMA) in acetone (15 ml)/DMSO (1 ml)at 50° C. for 24 h using N,N′-azobisisobutyronitrile (AIBN, 60 mg) asthe initiator as shown in Scheme 6. A tiny amount of 3-mercaptopropionicacid was used as a chain transfer agent. Typically the ratio ofcomonomers:initiator:solvent was kept constant at 8.8:0.4:90.8 wt %. Themixture was sealed under nitrogen in an ampoule and left to polymerizewith stirring at 50° C. for 24 h. The precipitated polymer was dissolvedin methanol, reprecipitated in 20× volume of ether and washed withether. Small molecular weight unreacted monomers and other impuritieswere separated from the polymeric conjugates by redissolving indistilled water or 50 mM Tris HCl buffer (pH7.2) and dialyzed againstdistilled water or 50 mM Tris HCl buffer (pH7.2) and subsequentlylyophilized to obtain the pure product.

Polymer-Gly-Phe-Leu-Gly-Docetaxel (SEQ ID NO: 1) using 2-APMA or 3-CPMAcan be prepared in a similar manner.

11) Polymer-Gly-Phe-Leu-Gly-Docetaxel/Gemcitabine (SEQ ID NO: 1)Preparation

HPMA, 2-APMA, 3-APMA, 2-CPMA, or 3-CPMA copolymer-docetaxel/gemcitabineconjugate are synthesized from the comonomers, by free radicalprecipitation copolymerization of the comonomers HPMA, 2-APMA, 3-APMA or3-CPMA (1.00 mmol), MA-GFLG-gemcitabine (SEQ ID NO: 1) (0.089 mmol) andMA-GFLG-docetaxel (SEQ ID NO: 1) (0.022 mmol) in acetone (2 ml) at 50°C. for 24 h using N,N′-azobisisobutyronitrile (AIBN, 10.8 mg) as theinitiator as shown in Scheme 6. Typically the ratio ofcomonomers:initiator:solvent is kept constant at 12.5:0.6:86.9 wt %. Themixture is sealed under nitrogen in an ampoule and left to polymerizewith stirring at 50° C. for 24 h. The precipitated polymer is dissolvedin methanol, reprecipitated in 20× volume of ether and washed withether. Small molecular weight unreacted monomers and other impuritiesare separated from the polymeric conjugates by redissolving in distilledwater and dialyzing against distilled water to remove the salts andsubsequently lyophilizing to obtain the pure product.

12) Synthesis of HPMA Copolymer-RGDfK-Drug Conjugate

Polymeric conjugates are synthesized in a two step procedure as outlinedin Scheme 7. In the first step the reactive HPMA, 2-APMA, 3-APMA 2-CPMA,or 3-CPMA copolymer-drug conjugates are synthesized by free radicalprecipitation copolymerization of comonomer HPMA, 2-APMA, 3-APMA 2-CPMA,or 3-CPMA, MA-GFLG-Docetaxel (SEQ ID NO: 1) and/or Gemcitabine andmethacryloylglycylglycine-p-nitrophenyl ester (MA-GG-ONp) comonomers inacetone/5% DMSO. The feed compositions of the comonomers are about89.34%, 0.66% and 10% respectively. N,N′-azobisisobutyronitrile (AIBN)is used as the initiator. Briefly comonomer HPMA, 2-APMA, 3-APMA 2-CPMA,or 3-CPMA (0.38 mmol), MA-GFLG-Docetaxel (SEQ ID NO: 1) and/orGemcitabine (0.0028 mmol) and MA-GG-ONp (0.0424 mmol) and AIBN (3.43 mg)are dissolved in 1 ml of acetone (5% DMSO). The ratio ofcomonomers:initiator:solvent is kept constant at 12.5:0.6:86.9 wt %. Themixture is sealed under nitrogen in an ampoule and left to polymerizewith stirring at 50° C. for 24 h. The precipitated polymer precursor isdissolved in methanol and reprecipitated in 20× volume of ether. Smallmolecular weight unreacted monomers and other impurities are separatedfrom the polymeric conjugates by redissolving in distilled water anddialyzing against distilled water to remove the salts and subsequentlylyophilizing to obtain the pure product. The ONp content of the polymeris determined spectrophometrically at 272 nm.

In the second step the targeting peptide such as RGDfK is conjugated topolymer precursors by an aminolysis reaction. Briefly HPMA, 2-APMA,3-APMA 2-CPMA, or 3-CPMA-(GFLG-Docetaxel and/or Gemcitabine (SEQ ID NO:1))-GG-ONp precursor (containing 0.02 mmol ONp groups) is dissolved in1.6 ml dry DMF (dried over 3 Å molecular sieves). RGDfK (0.03 mmol) isadded at 1.3 molar excess to that of the MA-GG-ONp content in thepolymeric precursor. The reaction is carried out under nitrogen for 24 hat room temperature. The reaction is terminated with 1-amino-2-propanol(0.02 mmol). The conjugate is dialyzed against deionized water andlyophilized.

13) Physicochemical Characterization of Polymer-Drug Conjugates

The weight average molecular weight (Mw) and polydispersity of thesynthesized polymer-drug conjugates were estimated by size-exclusionchromatography using a Superose 12 HR 10/30 column (AmershamBiosciences) on a Fast Protein Liquid Chromatography (FPLC) system(Amersham Biosciences). Samples at 1 mg/ml are eluted at a flow rate of0.4 ml/min using PBS as the elution solvent. The number averagemolecular weight (Mn), weight average molecular weight (Mw) andpolydispersity (n=Mw/Mn)) of the polymers were estimated from acalibration curve using polyHPMA or poly2-CPMA or poly3-APMA fractionsof known molecular weights. The drug content was obtained using AminoAcid Analysis (AIBio Tech., Richmond, Va.).

TABLE 1 Physicochemical characteristics of polymer-drug conjugates.‘GFLG’ is disclosed as SEQ ID NO: 1. Feed Monomer Composition Polymer(mole %) Characteristics MA- Drug Content Acry- GFLG- (mmole/g Mw Samplelate Drug polymer) (g/mole) Mw/Mn pHPMA- 97.5 2.5 0.104 10 kD 1.35GFLG-Docetaxel p2-CPMA- 97.5 2.5 0.0659 13 kD 1.24 GFLG-Docetaxelp3-APMA- 97.5 2.5 0.0446 10 kD 1.22 GFLG-Docetaxel

Example 2 Synthesis of Polymer-Drug A/Polymer-Drug B Conjugate (Scheme8)

MA-GFLG-ONp is prepared from MA-GFLG-OH (as shown above). TheMA-GFLG-Drug A or MA-GFLG-Drug B monomers can be prepared as outlinedabove. Poly(HPMA-co-MA-GFLG-ONp-MA-GFLG-Drug A) is prepared by freeradical polymerization of MA-GFLG-ONp, MA-GFLG-Drug A, and HPMA usingAIBN as an initiator. Poly(HPMA-co-MA-APMA-MA-GFLG-Drug B) is preparedby free radical polymerization of MA-GFLG-Drug B, HPMA and 3-APMA usingAIBN as an initiator. Subsequently,poly(HPMA-co-MA-GFLG-ONp-MA-GFLG-Drug A) is reacted withpoly(HPMA-co-MA-APMA-MA-GFLG-Drug B) to form the conjugate. ‘GFLG’ isdisclosed as SEQ ID NO: 1.

Example 3 Biological Tests Growth of Cancer Cell Lines

Cancer cell lines to determine the effect of polymer-drug conjugateswere obtained from the following sources: Human MDA-MB-231 (breast),HCT116 (colon) and PANC-1 (pancreas), from the American Type CultureCollection (ATCC) (Manassas, Va.). UMRC2 (kidney) from United StatesNational Cancer Institute (Bethesda, Md.). Cells were maintained inDulbecco's modified Eagle's medium (“DMEM”, Invitrogen) supplementedwith 10% FBS, P/S and 10 mM HEPES. All cells were incubated at 37° C.under humidified 5% CO₂.

2) In Vitro Cell Proliferation Assay Against Human Tumor Cell Lines

The growth inhibition assay of polymer-drug conjugates against humancancer cell lines was performed using the Sulforhodamine B (“SRB”)method (Skehan et al., J. National Cancer Institute, 82: 1107-1112(1990)). Briefly, exponentially growing cancer cells were seeded into a96-well plate at a density of 2−3×10³ cells/well and treated withcopolymer-drug conjugates the next day. Triplicate wells were used foreach treatment. Water was used as a control. The cells were incubatedwith copolymer-drug conjugates for 96 hours at 37° C. in a humidified 5%CO₂ atmosphere. After 96-hour incubation, cells were fixed with 10%trichloroacetic acid (“TCA”), incubated for 1 hour at 4° C., and washed3 times with tap water. Subsequently, cells were stained with 0.4%sulforhodamine B in 1% acetic acid for 30 minutes, washed 3 times with1% acetic acid, and air-dried again. After 5 minutes agitation in 10 mMTris solution, the absorbance of each well was measured at 530 nm usingBenchmark Plus Microplate reader (Bio-Rad Laboratories, Hercules,Calif.). The absorbance value provides a direct measure of the number oflive cells post-treatment with copolymer-drug conjugates.

To translate the OD₅₃₀ values into the number of live cells in eachwell, the OD₅₃₀ values were compared to those on standardOD₅₃₀-versus-cell number curves generated for each cell line. Thepercent survival was calculated using the formula:

% Survival=live cell number [test]/live cell number [control]×100

The IC₅₀ values were calculated by non-linear regression analysis.

TABLE 2 In-vitro cytotoxicity of docetaxel, pHPMA-GFLG-Docetaxel, p2-CPMA-GFLG-Docetaxel and p3-APMA-GFLG-Docetaxel against human cancer celllines. ‘GFLG’ is disclosed as SEQ ID NO: 1. IC₅₀ (μM) of drug equivalentDrugs Caki-1 MDA-MB-231 HCT116 Docetaxel 0.0010 0.0015 0.00058pHPMA-GFLG-Docetaxel 0.011 0.013 0.0056 p2-CPMA-GFLG-Docetaxel 0.0130.013 0.0056 p3-APMA-GFLG-Docetaxel 0.013 0.017 0.0090

Example 4 Ex Vivo Xenograft Study

In order to observe the inhibition of growth of tumor in an animalmodel, a nude mouse xenograft model were conducted utilizing polymerconjugated docetaxel or gemcitabine as described below.

HCT116 cell suspension (2×10⁶ cells in 0.1 ml of RPMI) were injectedsubcutaneously into the right flank of six-week-old male athymic mice(BALB/c nu/nu) on day 0. A sufficient number of mice were injected withHCT116 cell suspension so that tumors in a volume range as narrow aspossible were selected for the trial on the day of treatment initiation.Animals with tumors in the proper size range were assigned to varioustreatment groups and were injected with phosphate buffered saline (PBS)only or with a polymer-drug conjugate of the invention. All studymedications are given by intraperitoneal injections two times per weekstarting from day 5 and ending on day 32. To quantify tumor growth,three perpendicular diameters of the tumors are measured with calipersevery 3-5 days, and the body weight of the mice is monitored fortoxicity. The tumor volume is calculated using the formula: tumor volume(mm³)=(width)×(length)×(height)×π/6. Results are shown in FIG. 1.

As described herein, all embodiments or subcombinations may be used incombination with all other embodiments or subcombinations, unlessmutually exclusive.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described.

1-20. (canceled)
 21. A therapeutic composition comprising a firstpolymer carrier comprises (a) a first monomer selected from the groupconsisting of N-(3-aminopropyl)methacrylamide (3-APMA), andN-(2-aminopropyl)methacrylamide (2-APMA), and their combination; and (b)optionally a second monomer; and a first anticancer agent attached tothe first polymer carrier, optionally through a first linker; and atargeting ligand attached to the first polymer carrier, optionallythrough a second linker, wherein the targeting ligand is selected fromthe group consisting of RGDfK (arginine-glycine-asparticacid-D-phenylalanine-lysine oligopeptide), EPPT1 (YCAREPPTRTFAYWG, thatis, tyrosine-cysteine-alanine-arginine-glutamicacid-proline-proline-threonine-arginine-threonine-phenylalanine-alanine-tyrosine-tryptophane-glycineoligopeptide),and folate.
 22. The composition of claim 21, wherein the firstanticancer agent is attached to the first polymer carrier through thefirst linker, wherein the first anticancer agent is selected from thegroup consisting of docetaxel, gemcitabine, cisplatin, and theircombination.
 23. The composition of claim 21, wherein the first polymercarrier further comprises a second monomer selected from the groupconsisting of N-(2-hydroxypropyl)methacrylamide (HPMA),N-(2-carboxypropyl) methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA), an acrylamide, amethacrylamide, an acrylate, and a methacrylate.
 24. The composition ofclaim 21, further comprising an additional anticancer agent attached tothe first polymer carrier, optionally through a linker, wherein theadditional anticancer agent is different than the first anticanceragent, and wherein the additional anticancer agent is attached to thefirst monomer or if a second monomer is present, the additionalanticancer agent is attached to the first monomer or the second monomer.25. The composition of claim 21, wherein the targeting ligand isattached to the first monomer or, if a second monomer is present, thetargeting ligand is attached to the first monomer or the second monomer.26. The composition of claim 21, further comprising a second polymercarrier; and a second anticancer agent attached to the second polymercarrier, optionally through a linker; wherein the second polymer carriercomprises a second monomer selected from the group consisting ofN-(2-hydroxypropyl)methacrylamide (HPMA), N-(2-carboxypropyl)methacrylamide (2-CPMA), N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(3-aminopropyl)methacrylamide (3-APMA), andN-(2-aminopropyl)methacrylamide (2-APMA), and combinations, and whereinthe second anticancer agent is selected from the group consisting ofdocetaxel, gemcitabine, cisplatin, and their combination.
 27. Thecomposition of claim 21, wherein said first polymer carrier comprises(a) between about 5.0 and about 99.7 mol % of underivatized monomerunits, (b) between about 0.2 and about 20.0 mol % of derivatized monomerunits attached to the first anticancer agent; and (c) between about 0and about 94.8 mol % of derivatized monomer units attached to atargeting ligand.
 28. The composition of claim 27, wherein said firstpolymer carrier comprises between about 0.1 and about 94.8 mol % ofderivatized monomer units attached to the targeting ligand.
 29. Thecomposition of claim 21 having one or more linkers, wherein each of theone or more linkers is susceptible to cleavage by lysosomal enzymes. 30.The composition of claim 29, wherein the linker is selected from thegroup consisting of oligopeptide sequences, oligosaccharide sequencesand structures similar to those in nucleic acids.
 31. The composition ofclaim 30, wherein the linker is an oligopeptide sequence selected fromthe group consisting of Gly-Gly, Gly-Phe-Gly, Gly-Phe-Phe, Gly-Leu-Gly,Gly-Val-Ala, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Ala-Val-Ala,Gly-Phe-Leu-Gly (SEQ ID NO: 1), Gly-Phe-Phe-Leu (SEQ ID NO: 2),Gly-Leu-Leu-Gly (SEQ ID NO: 3), Gly-Phe-Tyr-Ala (SEQ ID NO: 4),Gly-Phe-Gly-Phe (SEQ ID NO: 5), Ala-Gly-Val-Phe (SEQ ID NO: 6),Gly-Phe-Phe-Gly (SEQ ID NO: 7), Gly-Phe-Leu-Gly-Phe (SEQ ID NO: 8), orGly-Gly-Phe-Leu-Gly-Phe (SEQ ID NO: 9).
 32. The composition of claim 31,wherein the oligopeptide sequence is Gly-Phe-Leu-Gly (SEQ ID NO: 1). 33.A pharmaceutical composition comprising the composition of claim 21 anda pharmaceutically acceptable carrier.
 34. A method of treating a canceror neoplastic disease comprising administering a therapeuticallyeffective amount of a composition according to claim
 21. 35. The methodof claim 34, wherein the cancer or neoplastic disease is selected fromthe group consisting of carcinomas, carcinomas of solid tissues,squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high gradegliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas,melanomas, adenomas, hypoxic tumours, myelomas, metastatic cancers,cervical dysplasias, anal dysplasias, severe dysplasias, hyperplasias,atypical hyperplasias, and neoplasias.
 36. A method of making atherapeutic composition comprising: reacting a first polymer carrierwith a first anticancer agent, optionally through a linker, to produce afirst conjugated drug monomer, wherein the first polymer carriercomprises a first monomer selected from the group consisting ofN-(3-aminopropyl)methacrylamide (3-APMA), andN-(2-aminopropyl)methacrylamide (2-APMA), and their combination;polymerizing the first conjugated drug monomers to form a polymer drugconjugate; and adding a targeting ligand to the polymer drug conjugate,wherein the targeting ligand is selected from the group consisting ofRGDfK (arginine-glycine-aspartic acid-D-phenylalanine-lysineoligopeptide), EPPT1 (YCAREPPTRTFAYWG, that is,tyrosine-cysteine-alanine-arginine-glutamicacid-proline-proline-threonine-arginine-threonine-phenylalanine-alanine-tyrosine-tryptophane-glycineoligopeptide),and folate.
 37. The method of claim 36, further comprising coupling thelinker with the first anticancer agent, wherein the linker is selectedfrom the group consisting of oligopeptide sequences, oligosaccharidesequences and structures similar to those in nucleic acids, and whereinthe linker is coupled to the first monomer molecule and wherein thefirst anticancer agent is selected from the group consisting ofdocetaxel, gemcitabine, cisplatin, and their combination.
 38. The methodof claim 36, wherein the first polymer carrier further comprises asecond monomer selected from the group consisting ofN-(2-hydroxypropyl)methacrylamide (HPMA), N-(2-carboxypropyl)methacrylamide (2-CPMA), N-(3-carboxypropyl)methacrylamide (3-CPMA), anacrylamide, a methacrylamide, an acrylate, and a methacrylate.
 39. Themethod of claim 36, further comprising reacting a second anticanceragent with the first polymer carrier, optionally through a linker,wherein the additional anticancer agent is different than the firstanticancer agent, and wherein the additional anticancer agent isattached to the first monomer or if a second monomer is present, theadditional anticancer agent is attached to the first monomer or thesecond monomer.
 40. The method of claim 36, further comprisingpolymerizing a second conjugated drug monomer with the first conjugateddrug monomer to form the polymer drug conjugate, wherein the secondconjugated drug monomer comprises a second monomer selected from thegroup consisting of N-(2-hydroxypropyl)methacrylamide (HPMA),N-(2-carboxypropyl) methacrylamide (2-CPMA),N-(3-carboxypropyl)methacrylamide (3-CPMA),N-(3-aminopropyl)methacrylamide (3-APMA), andN-(2-aminopropyl)methacrylamide (2-APMA), and combinations, and whereinthe second anticancer agent is selected from the group consisting ofdocetaxel, gemcitabine, cisplatin, and their combination.